bioRxiv preprint doi: https://doi.org/10.1101/492025; this version posted December 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. TOWARDS SPIDER GLUE:LONG-READ SCAFFOLDING FOR EXTREME LENGTH AND REPETITIOUS SILK FAMILY GENES AGSP1 AND AGSP2 WITH INSIGHTS INTO FUNCTIONAL ADAPTATION APREPRINT Sarah D. Stellwagen Rebecca L. Renberg Department of Biological Sciences General Technical Services University of Maryland Baltimore County Adelphi, MD 20783 Baltimore, MD 21250 [email protected] [email protected] December 19, 2018 ABSTRACT The aggregate gland glycoprotein glue coating the prey-capture threads of orb weaving and cobweb weaving spider webs is comprised of silk protein spidroins (spider fibroins) encoded by two members of the silk gene family. It functions to retain prey that make contact with the web, but differs from solid silk fibers as it is a viscoelastic, amorphic, wet adhesive that is responsive to environmental conditions. Most spidroins are extremely large, highly repetitive genes that are impossible to sequence using only short-read technology. We sequenced for the first time the complete genomic Aggregate Spidroin 1 (AgSp1) and Aggregate Spidroin 2 (AgSp2) glue genes of Argiope trifasciata by using error-prone long reads to scaffold for high accuracy short reads. The massive coding sequences are 42,270 bp (AgSp1) and 20,526 bp (AgSp2) in length, the largest silk genes currently described. The majority of the predicted amino acid sequence of AgSp1 consists of two similar but distinct motifs that are repeated ~40 times each, while AgSp2 contains ~48 repetitions of an AgSp1-similar motif, interspersed by regions high in glutamine. Comparisons of AgSp repetitive motifs from orb web and cobweb spiders show regions of strict conservation followed by striking diversification. Glues from these two spider families have evolved contrasting material properties in adhesion, extensibility, and elasticity, which we link to mechanisms established for related silk genes in the same family. Full-length aggregate spidroin sequences from diverse species with differing material characteristics will provide insights for designing tunable bio-inspired adhesives for a variety of unique purposes. Keywords spidroin · aggregate glue · long reads · full-length gene · spider silk · Argiope trifasciata 1 Introduction Spiders use a suite of remarkable silk and silk-derived materials for various applications during their life cycles, from wrapping prey and egg cases, to creating webs, lifelines, and prey capture glues. Aggregate gland glue is produced by spiders within the superfamily Araneoidea and functions to retain prey that get caught in a spider’s silken trap. This material is an adhesive glycoprotein (Tillinghast, 1981) that coats the capture spiral silk of orb web weavers, the silk trip lines of cobweb weavers, and the globule of glue at the end of the bolas of bolas spiders (Fig. 1). The glue is produced within a spider’s abdomen by two pairs of aggregate glands that terminate with valved spigots on the spinnerets (Sekiguchi, 1952). In orb weavers, each pair of semi-circle-shaped glue spigots surrounds a flagelliform silk spigot, which produces the supporting thread as the glue is concurrently extruded. The amorphous glue is deposited on the flagelliform threads as a continuous cylinder, however as the two coated threads merge to form a single strand, hygroscopic low molecular bioRxiv preprint doi: https://doi.org/10.1101/492025; this version posted December 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A PREPRINT -DECEMBER 19, 2018 mass compounds (LMMC) and salts absorb atmospheric moisture. Surface tension then causes the cylinder of glue to separate into equally spaced droplets (Fig. 1A)(Plateau, 1873; Boys, 1889; Edmonds and Vollrath, 1992). A single droplet contains a core of adhesive glycoprotein (Opell and Hendricks, 2010; Sahni et al., 2010) surrounded by an outer aqueous layer containing the LMMC and salts that retain water and lubricate the core of glue (Fischer and Brander, 1960; Anderson and Tillinghast, 1980; Tillinghast and Christenson, 1984; Townley et al., 1991; Opell et al., 2011, 2013; Sahni et al., 2014). Orb weaving spider glue acts as a viscoelastic solid, exhibiting viscous properties at more rapid extension rates, and elasticity at slow extension rates (Sahni et al., 2010). This allows the glue to adhere to fast-moving insects at interception, and retain insects while a spider travels to and subdues them. The stickiness of the glue droplets, however, is constrained by the force required to break the flagelliform support fibers, as glue that does not preemptively release results in damaged webs as an insect pulls free by breaking the support fibers (Agnarsson and Blackledge, 2009). Capture spiral threads also exhibit a suspension bridge mechanism, allowing forces to be distributed across multiple droplets and reducing thread peeling that would occur if only droplets on the ends of a contacted thread contributed to adhesion (Opell and Hendricks, 2007). Orb weaver glue responds instantly to humidity, which changes droplet volume and influences the performance of the glue (Opell et al., 2011; Sahni et al., 2011a). Orb weavers produce glue optimized for their habitat’s humidity, for example the glue of species from drier habitats becomes over-lubricated when exposed to higher humidity, while glue from species occupying wetter habitats becomes too viscous in drier conditions. Moreover, temperature and ultraviolet exposure also influence performance optima (Stellwagen et al., 2014, 2015). In contrast to orb weavers, cobweb weavers connect major ampullate silk threads from their webs to the ground, depositing aggregate glue on the lower portions to produce trip lines aimed at ambulatory prey (Fig. 1B). The trip lines, or ‘gumfoot’ threads, easily release from the ground, pulling insects into the air when accidentally intercepted, and the prey hang suspended until the spider can subdue them (Argintean et al., 2006). Unlike orb weaver glue, cobweb glue is considered a viscoelastic liquid, is resistant to changes in humidity, and is less extensible (Sahni et al., 2011a). Gumfoot droplets lack the glycoprotein core visible in those of orb weavers, and tend to flow together and coalesce rather than simply swelling when exposed to increased humidity (see figure 1 in Sahni et al. (2011a)). Cobweb aggregate gland pairs are morphologically distinct, and have likely diverged in function as well (Kovoor, 1977; Townley and Tillinghast, 2013; Clarke et al., 2017). Bolas spiders capture male moths using a single large glue droplet that hangs at the end of a silk strand (Fig. 1C). By mimicking female moth pheromones, the bolas spider lures the males to within striking range, and then swings their sticky snare until contact is made (Eberhard, 1977). The remarkable glue of these spiders is able to adhere in spite of a moth’s scales, which easily detach allowing them to escape other forms of predation (Sahni et al., 2011b). The aggregate glue of related moth specialist Cyrtarachne akirai Tanikawa, 2013 was recently characterized and was shown to have unique properties that allow the glue to spread and penetrate a moth’s scales (Diaz et al., 2018). The proteins that form spider silks and glues are from the same family termed spidroins (‘spider fibroins’) (Hinman and Lewis, 1992), and are often encoded by very long (>5kb), repetitive, single exon genes. Functional specialization of spidroin paralogs is associated with the evolutionary success of spiders (Shultz, 1987), particularly the central repetitive motifs that are responsible for the diversity of silk material properties (Gosline et al., 1999). The first full-length spidroin cDNAs were isolated from the orb weaving spider Argiope bruennichi (Scopoli, 1772) in 2006 (Zhao et al., 2006). The ~9 kb A. bruennichi cylindrical gland spidroins (CySp1 and CySp2; synonymous with tubiliform spidroins, TuSp), encode for silk that is used to wrap egg sacs. The gene that encodes for the most well known spidroin, major ampullate silk (more colloquially known as dragline silk), was fully sequenced from the black widow Latrodectus hesperus Chamberlin & Ivie (1935) genomic DNA the following year (Ayoub et al., 2007). To date, 19 large (>5kb), full-length, and classified spidroins (i.e., assigned to a specific silk gland source) have been described (Table 1). Several other smaller or unclassified silk gland genes have also been characterized (Babb et al., 2017). Initial evidence for aggregate glue gene sequences was derived from what were thought to be two full-length transcripts encoding the glue proteins of orb weaver Nephila clavipes (Linnaeus, 1767), and were named ASG1 (Aggregate Spider Glue; 1,221 bp) and ASG2 (2,145 bp) (Choresh et al., 2009). Several years later, it was determined that ASG1 expression is not unique to aggregate glands, and that the ASG2 sequence, while mostly correct, contained a short insertion (Collin et al., 2016). Collin et al. (2016) also discovered that the predicted ASG2 C-terminus aligns with other spidroins, and it was suggested ASG2 be renamed to AgSp1 (Aggregate Spidroin 1) to follow gene family convention, which we have adopted here. This study extended the known sequence to 2,821 bp, however the complete gene including the 5’ end encoding the predicted N-terminus still remained unidentified. During sequencing of the first orb weaving spider genome, which focused on describing spidroins from N. clavipes, the 5’ sequence for AgSp1 was discovered (Babb et al. (2017); called AgSp-c, following the study’s own spidroin naming scheme). While Babb et al. (2017) were unable to bridge a gap between the 5’ (7,660 bp) and 3’ (3,446 bp) ends of the gene, the known length of 2 bioRxiv preprint doi: https://doi.org/10.1101/492025; this version posted December 20, 2018.